The present invention concerns diagnostic means and methods in the field of coagulation testing. In particular, it relates to a method for determining an anticoagulant activity elicited by a first anticoagulant in a sample of a subject comprising measuring a first Factor Xa activity in a body fluid test sample of said subject, measuring a second Factor Xa activity in at least one calibrator sample comprising a predefined anticoagulation activity for a second anticoagulant, calculating a universal parameter for the anticoagulation activity comprised in the test sample based on the first and the second measured Factor Xa activities and comparing the said parameter for the anticoagulation activity with predefined ranges of expected anticoagulation activity for at least three anticoagulants. Further provided is a computer program code assisting the method as well as a system for carrying out the said method as well as a kit.
Any bigger organism has a blood circulation system, which brings oxygen and nutrients to the different organs, and disposes carbon dioxide and wastes. However for the blood circulation system to function, injuries in the blood vessels have to be closed rapidly and effectively. This function is fulfilled by the blood coagulation system, which is a complex mechanism which allows the blood to form platelet aggregates and fibrin gels, which are able to close vascular injuries.
However, blood clotting can not only lead to hemostasis, i.e. the closing of injuries in blood vessels, but also to thrombosis and embolism, i.e. the closure of blood vessels by blood clots. Thrombosis and embolism can have many manifestations, such as venous thrombosis in the legs, pulmonary embolism, myocardial infarction and stroke. The blood clotting system is therefore an important life-saving process, which can however also cause severe complications and even the death of the patient, if blood clotting closes vital blood vessels.
One mechanism involved in the blood coagulation system is the clotting factor cascade, which is a series of serine proteases, which become serially activated and ultimately lead to the formation of thrombin, the central enzyme of the blood clotting system. Thrombin is able to split fibrinogen to fibrin, which falls out, polymerizes into fibrin fibers, which form a fibrin clot. Thrombin is also activating co-factors, which accelerate its own generation (Factor V and Factor VIII), activates Factor XIII, a transglutaminase, which cross-links and thus stabilizes the fibrin clot, and thrombin is also a potent activator of the blood platelets.
As individuals become older and also accelerated by risk factors such as diabetes, obesity, smoking and genetic risk factors, there is an increasing risk for thrombotic events. Therefore drugs that inhibit the coagulation system have been developed, the so-called anticoagulants. One of the most successful classes of anticoagulant drugs are the inhibitors of Factor Xa, a serine protease which activates prothrombin to thrombin. The formation of Factor Xa is the step directly preceding the activation of thrombin.
Factor Xa is inhibited by several different drugs, such as low molecular weight heparin, Pentasaccharide, Rivaroxaban, Apixaban, and unfractionated heparin. These drugs have different structures, molecular weights, as well as mechanisms, but they share the common feature that they all lead to the inhibition of Factor Xa and therefore to a reduction in thrombin generation.
Most drugs directed against Factor Xa are generally very safe and do not require a routine monitoring of their effect in the clinical application. Still there are situations where the ability to measure the activity of these drugs is desirable: For example when the treating physician suspects that the patient might not reliably take his medication and wants to control the drug level, or if the patient has a disease that might lead to an accumulation of the drug in the circulation and therefore to bleeding complications, or in patients that are very old, in children or severely obese patients, or in patients experiencing complications during their anticoagulant therapy, i.e. bleeding or thrombosis, and the physicians want to elucidate the current anticoagulation status.
Two global assays are commonly used to measure the activity of the clotting factors: The prothrombin time (PT) and the activated partial thromboplastin time (aPTT). In both assays the clotting cascade is stimulated at its beginning (by tissue factor in the PT and by a contact activator in the aPTT) and following a series of enzymatic reactions thrombin is formed and the sample clots. The time between the start of the test and the clotting of the sample is the clotting time which is indicative of the activity of the clotting factors. However in both assays the formation of Factor Xa is only one step out of many and therefore the aPTT and PT have a low sensitivity for most inhibitors of Factor Xa and a poor quantification of the actual drug activity (see, e.g., EP 1 734 369 A1).
More specific methods for quantifying Factor Xa inhibition have been developed. Assays for measuring Factor Xa inhibition are also called “anti-Xa-tests” or “anti-Factor Xa-tests”. A common feature of such anti-Factor Xa-tests is that a sample is added to two reagents: One that contains Factor Xa and one that contains a peptide substrate which can be split by Factor Xa. Then the conversion of the peptide substrate by Factor Xa is recorded in the reaction solution. The peptide substrate comprises a certain amino acid sequence which allows it to be cleaved by Factor Xa, whereby the velocity of the conversion is proportional to the activity of Factor Xa in the sample. When the substrate is split by Factor Xa a signal reaction is mediated, which is measured by the analyzer, which performs the anti-Xa test. Usually a chemical group providing a detectable label (such as e.g. a chromogen or a fluorophor) is covalently bound to the peptide substrate. When such chromogenic peptide substrate is used a group which changes the colour of the solution is released, which can be measured photometrically. When a fluorogenic substrate is used a fluorescent group is released and when the reaction is measured electrochemically, the splitting of the substrate by Factor Xa results in the change of the ional structure of the reaction solution. The common feature of the different substrates is that in every case a signal reaction occurs in the sample, which is proportional to the concentration of Factor Xa in the sample and which is recorded by the analyzer.
Between the addition of the reagent containing Factor Xa to the sample and the addition of the reagent containing the substrate there can be an incubation step, in order to allow the Factor Xa inhibitor in the sample to inhibit Factor Xa. However, there are also assays with no such incubation step. Optionally, also other substances can be added which influence the specificity of the assay, such as dextrane sulfate or antithrombin.
The result of the assay is then the change of absorbance, or the rate of change of absorbance, (or fluorescence or any other signal reaction used). For simplicity it is assumed that the signal reaction is the change in absorbance expressed in mE (milli units of extinction).
As known in the art usually, using this absorbance the anticoagulant concentration was calculated using a calibration curve. This procedure was developed when only 2 classes of inhibitors to Factor Xa were therapeutically applied, namely unfractionated heparin (UFH) and low molecular weight heparin (LMWH). Typically, a separate calibration curve was performed with calibrators, which contain increasing doses of LMWH or UFH, and following the measurement of these calibrators the analyzer determines the calibration curve to calculate the anticoagulant concentration from the absorbance values determined with the plasma samples.
In the meantime, several additional Factor Xa inhibitors have been introduced into the clinical practice and calibrators as well as controls for e.g., Rivaroxaban, pentasaccharide, Danaparoid are available.
In any larger hospital, today, many different anticoagulants are simultaneously used. Some patients may receive LMWH for prophylaxis of deep venous thrombosis (DVT) during hospitalization, others may receive Rivaroxaban for the prophylaxis of stroke caused by atrial fibrillation, other patients receive Pentasaccharide for the prevention of DVT in hip replacement surgery, and intensive care unit (ICU) patients may receive therapy with unfractionated heparin.
The clinical process, typically, is as follows: Assistant personal (e.g., a phlebotomist, a nurse or one of the younger medical practitioners) prepares the blood collection tubes for the blood collection and the fills out the respective order forms that specify which assays have to be performed. The blood is collected and transferred to the laboratory together with the order form. If a test for LMWH, UFH, Rivaroxaban or Apixaban has been ordered an anti-Factor Xa test is performed, and the concentration of the anticoagulant is calculated based on the respective calibration curve. This concentration is then transferred to the treating physicians via the laboratory information system (LIS), which is usually available via intranet, or via a fax, letter or other means of information.
However, this procedure for ordering the assays, calibration, and result expression has several shortcomings. These limitations affect the efficiency of the process, but also impose medical risks.
For example, the need to use several sets of calibrators and controls for a single diagnostic method (the anti-Factor Xa-test) is cumbersome, expensive and adds complexity in the laboratory workup. If one imagines that every laboratory test required several different calibrations, controls, and proficiency testing procedures, one can imagine that this would add a great burden in the effort involved with laboratory analysis and also on the costs of a laboratory.
In addition risks are involved, which can be highlighted by the following example: As mentioned previously the order forms for blood tests are usually filled out not by the treating physicians themselves, but by assistant personnel having a lower medical education level or experience (nurses, phlebotomists, young doctors). If now, inadvertently, assistant personal orders a “LMWH” test, while the patient receives Rivaroxaban, the laboratory will report a LMWH concentration, even though the patient might have never been treated with this drug. This means that the expression of the laboratory result is more specific than the analytical method itself. What is measured is Factor Xa inhibition. However, what is reported is the concentration of a particular drug. In another scenario, one may assume that a physician evaluating a patient's laboratory values would see a drop in the platelet count and at the same time acertain LMWH concentration. The said physician could therefore misinterpret this result as a heparin induced thrombocytopenia. The problem is not only that a wrong order is propagated through the process, but the wrong information at the beginning is up-valued through the chain. If a nurse told the physician that the patient has received LMWH, the physician might double check this information. However if the physician receives a “LMWH concentration” in an official report from his laboratory, with the signature of the responsible laboratory staff on it, he will assume that this information is correct.
Therefore the current diagnostic procedure involves a very early selection of the calibration procedure which will be applied much later in the diagnostic process, wrong selections of the drug to be calibrated with are propagated throughout the entire diagnostic process and up-valued. The diagnostic procedure involving a generic measuring step (i.e. Factor Xa inhibition) and a calibration step (against the respective anticoagulant) is not transparent to the user. The calibration procedure is rigid, i.e. once the result has been reported, it is not possible to change the calibration even if the wrong drug has been selected earlier in the process (Favaloro 2011, Pathology, December; 43(7):682-92; Tripodi 2013, Clin Chem, February; 59(2):353-62; and Gehrie 2012, Am J Hematol., February; 87(2):194-6).
The technical problem underlying the present invention can be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.